Autoimmunity
Abstract
The individual contribution of specific myeloid subsets such as CD1c+ conventional dendritic cells (cDC) to perpetuation of Rheumatoid Arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDCs in RA patients and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional and functional characteristics of CD1c+ and CD141+ cDCs and monocytes from the blood and synovial fluid of RA patients. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC associated with the presence of this DC subset in the synovial membrane in RA patients. Moreover, synovial CD1c+ cDCs are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFNγ+IL-17+ CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ Receptors and NLRC4 as a new potential molecular mechanism mediating pathogenic activation, CD64 upregulation and functional specialization of CD1c+ cDCs in response to dsDNA-IgG in RA patients.
Authors
Cristina Delgado-Arévalo, Marta Calvet-Mirabent, Ana Triguero-Martinez, Enrique Vázquez de Luis, Alberto Benguría-Filippini, Raquel Largo, Diego Calzada-Fraile, Olga Popova, Ildefonso Sánchez-Cerrillo, Ilya Tsukalov, Roberto Moreno-Vellisca, Hortensia de la Fuente, Gabriel Herrero-Beaumont, Almudena R. Ramiro, Francisco Sánchez-Madrid, Santos Castañeda, Ana Dopazo, Isidoro González-Álvaro, Enrique Martin-Gayo
Abstract
Activation of Toll-like receptor 4 (TLR4) by its cognate damage-associated endogenous ligands (DAMPs) elicits potent profibrotic effects and myofibroblasts activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP-TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 KDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other (r=0.54, p=0.0062). Expression of RP105 was negatively associated with myofibroblast differentiation in SSc (r=-0.53). Importantly, RP105-TLR4 association was reduced while TLR4- TLR4 showed strong association in SSc fibroblasts as shown by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we identify RP105-MD1 as a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.
Authors
Wenxia Wang, Swarna Bale, Bharath Yalavarthi, Priyanka Verma, Pei-Suen Tsou, Ken M. Calderone, Dibyendu Bhattacharyya, Gary J. Fisher, John Varga, Swati Bhattacharyya
Abstract
Biased agonism is a frontier field in G-protein coupled receptor (GPCR) research. Acquired hypocalciuric hypercalcemia (AHH) is a rare disease caused by calcium-sensing receptor (CaSR) autoantibodies, to date, showing either simple blocking or biased properties (i.e., stimulatory or blocking effects on different downstream signaling pathways). This emphasizes the importance of the Gi/o (pertussis toxin-sensitive G proteins, whose βγ subunits activate multiple signals including ERK1/2) in regulating PTH secretion. We here describe three patients with symptomatic AHH that shared characteristics with the two cases we previously reported as follows: [1] aged (between 74-87 years at diagnosis); [2] male; [3] unexpectedly showed no other autoimmune diseases; [4] showed spontaneously fluctuating calcium levels from approximately normal to near fatally high ranges; [5] acute exacerbations could be successfully treated with prednisolone and/or calcimimetics; [6] the presence of CaSR autoantibodies that operated as biased allosteric modulators of CaSR; and that [7] were likely to be conformational (i.e., recognizing and thereby stabilizing a unique active conformation of CaSR that activates Gq/11, activating phosphatidylinositol turnover, but not Gi/o). Our observations with these prominent commonalities may provide new insights into the phenotype and characteristics of AHH and the mechanisms by which the biased agonism of GPCRs operate.
Authors
Noriko Makita, Junichiro Sato, Katsunori Manaka, Kimiko Akahane, Takahiro Ito, Hajime Yamazaki, Akira Mizoguchi, Yusuke Hikima, Hirofumi Horikoshi, Masaomi Nangaku, Taroh Iiri
Abstract
STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αβ T cell–dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ–induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-β, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.
Authors
W. Alexander Stinson, Cathrine A. Miner, Fang R. Zhao, Annena Jane Lundgren, Subhajit Poddar, Jonathan J. Miner
Abstract
The origin and mechanisms of autoantigen generation in systemic lupus erythematosus (SLE) are poorly understood. Here, we identified SLE neutrophils activated in vivo by interferon as a prominent source of Ro52/TRIM21 (hereafter Ro52), a critical autoantigen historically thought to be primarily generated by keratinocytes in SLE. Different to mononuclear cells and keratinocytes, SLE neutrophils are enriched in several unique Ro52 species containing a core sequence encoded by exon-4 (Ro52Ex4) in TRIM21. Ro52Ex4 is the main target of anti-Ro52 antibodies and is found in two Ro52 variants (Ro52α and a novel isoform termed Ro52γ) upregulated in SLE neutrophils. Further analysis of Ro52γ revealed a new subset of autoantibodies against a unique C-terminal domain (Ro52γCT) generated from a frameshift due to the lack of exon-6 in Ro52γ. Antibodies to Ro52Ex4 and Ro52γCT distinguish SLE patient subsets characterized by distinct clinical, laboratory, treatment and transcriptional profiles, which are not discerned by the “classical” anti-Ro52 antibodies. Together, these studies uncover interferon-activated neutrophils as a key source of unique immunogenic forms of Ro52 in SLE. Moreover, the finding of Ro52Ex4 and Ro52γCT as core targets of anti-Ro52 antibodies focus interest on Ro52γ as the potential isoform toward which immunological tolerance is initially lost in SLE.
Authors
Eduardo Gomez-Bañuelos, M. Javad Wahadat, Jessica Li, Merlin Paz, Brendan Antiochos, Alessandra Ida Celia, Victoria Andrade, Dylan P. Ferris, Daniel W. Goldman, Erika Darrah, Michelle Petri, Felipe Andrade
Abstract
T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep-sequenced the TCR beta chain (TCRβ) repertoires from longitudinal peripheral blood DNA samples at four time points beginning early in life (median age of 1.4 years) from children that progressed to T1D (n=29) and age/sex-matched islet autoantibody negative controls (n=25). From 53 million TCRβ sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independent of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and separately those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCRβ sequences are more common and frequent in number as the disease progressed in those who developed T1D compared to genetically at-risk non-diabetic children, which was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCRβ repertoires from a cohort of new-onset T1D patients (n=143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.
Authors
Angela M. Mitchell, Erin E. Baschal, Kristen A. McDaniel, Kimber M. Simmons, Laura Pyle, Kathleen Waugh, Andrea K. Steck, Liping Yu, Peter A. Gottlieb, Marian J. Rewers, Maki Nakayama, Aaron W. Michels
Abstract
Cub domain-containing protein 1 (CDCP1) is a surface protein highly expressed on the surface of many cancer cells, however, the distribution of CDCP1 in normal tissues and its potential roles in non-tumor cells are poorly understood. We previously reported that CDCP1 interacts with CD6, a surface marker of T cells, suggesting that it is a novel immunoregulator, but the physiological significance of the newly discovered CDCP1-CD6 interaction remains unclear. In this report, we found that CDCP1 is present on both human and mouse retinal pigmented epithelial cells (RPEs), a component of the blood-retina barrier (BRB), using a new anti-CDCP1 monoclonal antibody that we developed. CDCP1 knockout (KO) mice on two different genetic backgrounds both developed significantly attenuated retinal T cell infiltration and uveitis after adoptive transfer of pre-activated pathogenic T cells in a model of autoimmune uveitis. We also found that tight junctions were severely disrupted with infiltrating T cells detected in the RPE flat mounts prepared from the WT but not CDCP1 KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE was upregulated by IFNγ in vitro and after EAU induction in vivo. CD6 stimulation induced significantly increased RPE barrier permeability of WT, but not CDCP1 knockdown (KD) RPE, and activated T cells migrated through the WT RPE monolayes more efficiently than the CDCP1 KD RPE monolayers. In addition, CD6 stimulation of WT, but not the CDCP1 KD RPEs, induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPEs interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.
Authors
Lingjun Zhang, Nozha Borjini, Yu Lun, Sweta Parab, Gospel Enyindah-Asonye, Rupesh Singh, Brent A. Bell, Vera L. Bonilha, Andrei I. Ivanov, David A. Fox, Rachel R. Caspi, Feng Lin
Abstract
Checkpoint inhibitors (CPIs) targeting programmed death-1(PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications including CPI-induced diabetes (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti-PD-L1 but not anti-CTLA-4 induces diabetes rapidly. RNA sequencing revealed that cytolytic IFNγ+ CD8+ T cells infiltrated islets with anti-PD-L1. Changes in β cells were predominantly driven by IFNγ and TNFα and included induction of a potentially novel β cell population with transcriptional changes suggesting dedifferentiation. IFNγ increased checkpoint ligand expression and activated apoptosis pathways in human β cells in vitro. Treatment with anti-IFNγ and anti-TNFα prevented CPI-DM in anti-PD-L1 treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway result in transcriptional changes in β cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.
Authors
Ana Luisa Perdigoto, Songyan Deng, Katherine C. Du, Manik Kuchroo, Daniel B. Burkhardt, Alexander Tong, Gary Israel, Marie E. Robert, Stuart P. Weisberg, Nancy Kirkiles-Smith, Angeliki M. Stamatouli, Harriet M. Kluger, Zoe Quandt, Arabella Young, Mei-Ling Yang, Mark J. Mamula, Jordan S. Pober, Mark S. Anderson, Smita Krishnaswamy, Kevan C. Herold
Abstract
Gut microbiota (GM) dysbiosis is associated with inflammatory bowel diseases and also with cardiometabolic, neurologic, and autoimmune diseases. GM composition has a direct effect on the immune system, and vice versa, and particularly on regulatory T cell (Treg) homeostasis. Low-dose interleukin-2 (IL-2LD) stimulates Tregs and is a promising treatment for autoimmune and inflammatory diseases. We aimed to evaluate the impacts of IL-2LD on GM, and correlatively on the immune system. We used 16S ribosomal RNA profiling and metagenomics to characterize GM of mice and humans treated or not with IL-2LD. We performed faecal microbiota transplantation (FMT) from IL-2LD-treated to naïve recipient mice and evaluated its effects in models of gut inflammation and diabetes. IL-2LD markedly affects GM composition in mice and humans. Transfer of an IL-2-tuned microbiota by FMT protected C57BL/6J mice from dextran sulphate sodium-induced colitis and prevented diabetes in NOD mice. Metagenomic analyses highlighted a role for several species impacted by IL-2LD and for microbial pathways involved in the biosynthesis of amino acids, short-chain fatty acids, and L-arginine. Our results demonstrate that IL-2LD induces changes in GM that are involved in the immunoregulatory effects of IL-2LD and suggest a cross-talk between Tregs and GM. These results provide novel insights for understanding the mode of action of Treg-directed therapies.
Authors
Nicolas Tchitchek, Otriv Nguekap Tchoumba, Gabriel Pires, Sarah Dandou, Julien Campagne, Guillaume Churlaud, Gwladys Fourcade, Thomas W. Hoffmann, Francesco Strozzi, Camille Gaal, Christophe Bonny, Emmanuelle Le Chatelier, Stanislav Dusko Erlich, Harry Sokol, David Klatzmann
Abstract
Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a post-translational regulator of the low-density lipoprotein receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-sequencing-based variant discovery, we identified a novel psoriasis susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C>T). This finding was verified in independently acquired genomic and RNA-sequencing datasets. Single-cell RNA-sequencing (scRNA-seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-seq and immunohistochemistry demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL-36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression including, IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C>T was associated with lower PCSK9 expression and higher IL36G expression, when compared to heterozygous skin or cell lines. Together these results support PCSK9 as a novel psoriasis susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.
Authors
Alexander Merleev, Antonio Ji-Xu, Atrin Toussi, Lam C. Tsoi, Stephanie T. Le, Guillaume Luxardi, Xianying Xing, Rachael Wasikowski, William Liakos, Marie-Charlotte Brüggen, James T. Elder, Iannis E. Adamopoulos, Yoshihiro Izumiya, Annie Riera-Leal, Qinyuan Li, Nikolay Yu Kuzminykh, Amanda Kirane, Alina I. Marusina, Johann E. Gudjonsson, Emanual Maverakis
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